A study of the influence of charged residues on β-hairpin formation by nuclear magnetic resonance and molecular dynamics

Abstract

Chain reversals are often nucleation sites in protein folding. The β-hairpins of FBP28 WW domain and IgG are stable and have been proved to initiate the folding and are, therefore, suitable for studying the influence of charged residues on β-hairpin conformation. In this paper, we carried out NMR examination of the conformations in solution of two fragments from the FPB28 protein (PDB code: 1E0L) (N-terminal part) namely KTADGKT-NH2 (1E0L 12-18, D7) and YKTADGKTY-NH2 (1E0L 11-19, D9), one from the B3 domain of the protein G (PDB code: 1IGD), namely DDATKT-NH2 (1IGD 51-56) (Dag1), and three variants of Dag1 peptide: DVATKT-NH2 (Dag2), OVATKT-NH2 (Dag3) and KVATKT-NH2 (Dag4), respectively, in which the original charged residue were replaced with non-polar residues or modified charged residues. It was found that both the D7 and D9 peptides form a large fraction bent conformations. However, no hydrophobic contacts between the terminal Tyr residues of D9 occur, which suggests that the presence of a pair of like-charged residues stabilizes chain reversal. Conversely, only the Dag1 and Dag2 peptides exhibit some chain reversal; replacing the second aspartic-acid residue with a valine and the first one with a basic residue results in a nearly extended conformation. These results suggest that basic residues farther away in sequence can result in stabilization of chain reversal owing to screening of the non-polar core. Conversely, smaller distance in sequence prohibits this screening, while the presence oppositely-charged residues can stabilize a turn because of salt-bridge formation.

Fig. 1

a NMR structure [44], of the FBP28 WW domain (PDB code: 1E0L); the boxed fragments of the FBP28 WW domain: FPB28 (12-18) D7, and FPB28 (11-19) D9 were synthesized and examined. b X-ray structure [45] of the protein G (PDB code: 1IGD) derived from Streptococcus—B3 domain of immunoglobulin binding protein; the boxed fragment of the 1IGD (51-56) Dag1 and its mutants were synthesized and examined

Fig. 2

The ROE effects and the values of the vicinal ³J_HN/Hα coupling constants for D7 (a), D9 (b), Dag1 (c), Dag2 (d), Dag3 (e) and Dag4 (f) peptide

Fig. 3

The main family(s) clustered by using the MOLMOL program (hierarchical minimal spanning tree method) [–40] and the most representative conformations for D7 (a), D9 (b), Dag1 (c), Dag2 (d), Dag3 (e) and Dag4 (f) peptide

Fig. 4

The structural alignment between the most representative conformation of a D7, b D9 peptides with the corresponding fragment of FBP28 WW structure and c Dag1, d Dag2, e Dag3, f Dag4 peptides with the corresponding fragment of 1IGD protein structure